Manual Hemofilie

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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY

Neonatal gene transfer with a retroviral vector results in tolerance to humanfactor IX in mice and dogs

Jun Zhang, Lingfei Xu, Mark E. Haskins, and Katherine Parker Ponder

The effect of neonatal gene transfer on

antibody formation was determined us-

ing a retroviral vector (RV) expressing

human factor IX (hFIX). Normal mice from

different strains injected intravenously

with RV as newborns achieved therapeu-

tic levels of hFIX without antibody produc-

tion and were tolerant as adults to chal-

lenge with hFIX. Neonatal hemophilia B

mice that received different amounts of

RV achieved stable and dose-related ex-

pression of hFIX without anti-hFIX anti-

body formation. After protein challenge,

antibody formation was markedly re-

duced for animals that expressed hFIX at

levels higher than 14 ng/mL (0.3% of

normal). However, antibodies developed

for animals that received the lowest dose

of RV and expressed hFIX at approxi-

mately 2 ng/mL before protein challenge.

In dogs, neonatal injection of a high dose

of RVresulted in 500 ng/mL hFIX inplasma

without antibody formation. We conclude

that neonatal gene transfer with RV does

not induce antibody responses to hFIX in

mice or dogs and that mice achieving

levels greater than 3 1010 M hFIX are

usually tolerant to protein injection as

adults. Low-dose gene therapy or fre-

quent protein injections in the neonatal

period might induce tolerance to subse-

quent injections of protein with a low risk

for adverse effects. (Blood. 2004;103:

143-151)

2004 by The American Society of Hematology

IntroductionHemophilia B (HB) is an X-linked disorder caused by deficient

factor IX (FIX) activity that affects 1:30 000 males.1 Although gene

therapy can result in therapeutic levels of FIX in blood by

achieving continuous secretion of the 50-kDa FIX protein,2 anti-

body responses have occurred. Antibodies can reduce the coagula-

tion function (referred to as inhibitors) or increase the clearance of

protein from blood.

Anti-FIX antibodies often occur after gene transfer to adult

immunocompetent mice. Antibodies developed after intramuscular

(IM) injection of AAV23-8 or adenoviral9 vectors, intraperitoneal

(IP) injection of transduced fibroblasts, 10 intravenous (IV) injec-

tion of a retroviral vector (RV), 11 or IV injection of adenoviral

vectors in most strains except C57BL/6.12-15 Liver-restricted expres-sion may reduce the chance of antibody formation because IV

injection of AAV vectors, which are expressed primarily in the

liver, failed to induce antibodies,3,4 though antibodies developed

with varying frequency in other reports.16-18 Liver-restricted expres-

sion from an adenoviral vector may reduce antibody develop-

ment,19,20 though differences in the level of expression observed

with different vectors may affect the result. High expression is less

likely than low expression to induce antibody formation after the

delivery of AAV vectors to liver18 or muscle.8

Anti-FIX antibodies can also develop after gene transfer to

adult large animals. Anti-FIX antibodies developed after IM

injection of human FIX (hFIX)expressing plasmid21 or AAV22

vectors in dogs or a canine FIX (cFIX)expressing AAV vector inHB dogs from Auburn, which have a frameshift mutation and often

develop inhibitors.23 However, anti-cFIX antibodies did not de-

velop in an Auburn dog that received cyclophosphamide (Cytoxan)

before IM injection of an AAV vector,24 and they only developed in

1 of 3 Auburn dogs that expressed an AAV vector in the liver.25 The

Chapel Hill HB colony has a missense mutation and usually does

not produce inhibitors to cFIX.26 In the Chapel Hill dogs, anti-cFIX

antibodies did not develop after liver-directed gene therapy with

retroviral27 or AAV17,25,28 vectors, and they were stable in only 1 of

9 dogs after IM injection of an AAV vector.29-31 In Rhesus

macaques, antibodies to hFIX developed in 3 of 3 animals after IV

injection of an adenoviral vector32 and in 1 of 5 animals after

liver-directed, AAV-mediated gene transfer.33 Anti-hFIX antibodies

did not develop in any of the humans who received muscle-directed

AAV vector-mediated gene therapy.34 However, these patients wereat low risk for antibody formation because they had prior exposure

to hFIX without inhibitor development. Thus, inhibitor formation

remains a concern for gene therapy approaches in humans,

particularly those with null mutations.

Inhibitors also developed in approximately 3% of HB pa-

tients,35 but eradication with high doses of hFIX is expensive

and not always successful.36 Identifying a method to prevent

inhibitor formation after protein infusion would thus be an

important advance.

Our hypothesis was that gene transfer into newborns with

immature immune systems37,38 might prevent inhibitor formation.

This could involve a high dose to achieve fully therapeutic levels of

expression. Alternatively, low-dose gene therapy with subtherapeu-tic levels might induce tolerance to protein infusions with less

chance for adverse effects. We recently reported that neonatal gene

From the Departments of Internal Medicine and Biochemistry and Molecular

Biophysics, Washington University School of Medicine, St Louis, MO; and the

Department of Pathobiology, University of Pennsylvania School of Veterinary

Medicine, Philadelphia.

SubmittedJuly 1, 2003; accepted September3, 2003. Prepublishedonlineas Blood

First Edition Paper, September 11, 2003; DOI 10.1182/blood-2003-06-2181.

Supported by the National Institutes of Health grants DK48028 (K.P.P.) and

RR02512 (M.E.H.).

Reprints: Katherine P. Ponder, Department of Internal Medicine, Washington

University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110; e-mail:

[email protected].

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked advertisement in accordance with 18 U.S.C. section 1734.

2004 by The American Society of Hematology

143BLOOD, 1 JANUARY 2004 VOLUME 103, NUMBER 1


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transfer with an RV did not induce antibody formation to cFIX in

mice or HB dogs from Chapel Hill.11 We now examine the effect of

neonatal delivery of an RV expressing the immunogenic hFIX

protein. We conclude that neonatal gene transfer does not induce

antibody formation in mice or dogs and that most mice are tolerized

to subsequent infusions of protein.

Materials and methods

Reagents

Reagents were obtained from Sigma Chemical (St Louis, MO) unless

otherwise stated.

Retroviral vector construction

A 1.5-kb hFIX cDNA39 with an optimal Kozak sequence40 and 48 nt of

3-untranslated sequence was used. The hFIX cDNA was blunt-end ligated

into the NotI site of hAAT-WPRE-76741 to generate hAAT-hFIX-WPRE.

Generation of an amphotropic GPAM12-based42 packaging cell line and

large-scale production of the RV41 were as described previously. Titer in

transducing unit (TU) per milliliter was determined by immunostaining

after freezing once. Two days after NIH3T3 cells were infected, cells were

fixed with formalin for 20 minutes at room temperature (RT) and were

permeabilized for 10 minutes with methanol. Blocking buffer (Tris-

buffered saline [40 mM Tris-HCl, 150 mM NaCl, pH 7.4] with 5% nonfat

dry milk [TBS-milk; Schnucks Grocery, St Louis, MO]) was added at RT

for 1 hour, and wells were incubated with a goat anti-hFIX antibody

(GAFIX-AP; Enzyme Research Laboratories, South Bend, IN) at a 1:200

dilution for 1.5 hours. Cells were washed with TBS and then were incubated

with a mouse anti-goat/sheep immunoglobulin G (IgG) antibody at a 1:100

dilution at RT for 1.5 hours. Staining was developed with 3,3-

diamobenzidine.43 The RV had fewer than 10 copies of replication-

competent retrovirus by a vector rescue assay.41 Polybrene was added (final

concentration, 8 g/mL) before injection.

Animal procedures

National Institutes of Health and United States Department of Agriculture

guidelines for the care and use of animals were followed. Inbred BALB/

cByJ (referred to as BALB/c), C3H/HeJ (referred to as C3H), C;129S-

Cd1tm1Gru (these are CD1 deficient and lack natural killer function, but have

normal TH2 cell help and are referred to as BALB/c:129S), and C57BL/6J

(referred to as C57BL/6) mice were obtained from the Jackson Laboratory

(Bar Harbor, ME). HB mice were in a mixed 129SC57BL/6 back-

ground.44 Newborn mice were injected intravenously through the temporal

vein with 100 L RV at 2 to 3 days after birth. For protein challenge,

animals were injected IP with 0.6 international units (IU) hFIX (BeneFix,

specific activity 270 IU/mg; Wyeth Pharmaceutical, Cambridge, MA) in

300 L phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10.1

mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), which represented approxi-mately 30 IU/kg. Some mice were injected IP with 0.6 IU BeneFix in 200

L adjuvant RIBI MPLTDM emulsion (Corixa, Hamilton, MT), which

contains 0.5 mg/mL monophosphoryl lipid A, 0.5 mg/mL synthetic

trehalose dicorynomycolate, 2% squalene, and 0.2% Tween 80. Plasma was

collected through a nonheparinized capillary tube and was mixed with 0.1

vol of 3.2% sodium citrate.

Phenotypically normal puppies were identified by polymerase chain

reaction (PCR) analysis of blood samples after breeding mucopolysacchari-

dosis VII dogs from the University of Pennsylvania colony.45 At 2 or 3 days

after birth, 5 mL RV was injected as a single IV dose over 2 minutes.

Immunoassay for hFIX

Enzyme-linked immunosorbent assay (ELISA) plates were coated with

mouse monoclonal anti-hFIX antibody (HIX-1, F2645) at a 1:500 dilutionin PBS. Wells were blocked overnight with TBS-milk and then washed 6

times with TBS with 0.05% Tween 20 (TBS-Tween) after this and

subsequent steps. Samples were diluted in TBS-milk to give values on the

linear portion of the standard curve and were incubated at 37 C for 2 hours.

A horseradish peroxidase (HRP)conjugated goat anti-hFIX antibody

(GAFIX-APHRP; Enzyme Research Laboratories, South Bend, IN) at a

1:500 dilution was incubated for 2 hours at 37C, and the assay was

developed with 3,3, 5,5-tetramethylbenzidine. Standards were dilutions of

purified hFIX (Calbiochem, San Diego, CA).

Anti-hFIX IgG antibody assays

ELISA plates were coated with 5 g/mL purified hFIX (Calbiochem) in

PBS and were blocked with TBS-milk. Samples diluted 1:100 or higher in

TBS-milk were incubated overnight at 4C. For samples from mice, an

HRP-conjugated goat anti-mouse IgG that recognizes all subclasses of IgG

(Roche Molecular Biochemicals, Indianapolis, IN) was added at a 1:200

dilution at 37C for 2 hours, and the plate was developed with 3,3,

5,5-tetramethylbenzidine. For each assay, standards with 2 g/mL or less

mouse IgG with a normal mixture of subtypes (no. 1-5381; Sigma

Chemical, St Louis, MO) was used to calculate the relative amount of

antibody in milligrams per milliliter. The titer was the highest dilution at

which the optical density (OD) for a sample captured with hFIX was at least

twice the background OD for the same sample captured with a PBS-coated

well. For dog samples, an HRP-coupled sheep anti-canine IgG (Serotec,

Raleigh, NC) was added at a 1:500 dilution. Standards were dilutions of dog

plasma containing 3.5 g/mL or less of dog IgG (RS10-105; Bethyl

Laboratories, Montgomery, TX).

Bethesda assay

Samples were heat inactivated at 56C for 60 minutes. For mouse samples,

10 L mouse plasma, 30 L PBS, and 10 L normal human plasma

(George King Biomedical, Overland, KS) were incubated for 2 hours at

37C. Fifty microliters hFIX-deficient human plasma was added, and

activated partial thromboplastin time (aPTT) assay was performed.11

Coagulation times were compared with standards containing 0 to 10 L

normal human plasma, 10 to 0 L hFIX-deficient plasma, 10 L

heat-inactivated normal mouse plasma, and 30 L PBS that were preincu-

bated for 2 hours, after which aPTT was performed with 50 L hFIX-

deficient human plasma. The dilution factor was considered to be 1 if 10L

undiluted mouse plasma was used. If necessary, samples were diluted in

PBS, and values were compared with a standard curve with the same

amount of normal mouse plasma. One Bethesda unit (BU) per milliliter

inhibits 50% of the coagulation activity, and the limit of sensitivity was 1

BU/mL. Samples from dogs were assayed in a similar fashion except that

10 L heat-inactivated dog plasma was used instead of mouse plasma for

samples and standards.

Results

Generation of an RV-expressing hFIX

The goal of this project was to study immune responses after

neonatal gene transfer of RV. The hFIX cDNA was used because

mice and dogs usually make antibodies to the human protein, and

reagents are available to characterize the response. The Moloney

murine leukemia virus-based RV vector also contained the human

1-antitrypsin promoter and the woodchuck hepatitis virus post-

transcriptional regulatory element (Figure 1A). The titer of the

concentrated RV varied from 1.8 to 3.5 108 TU/mL.

Neonatal gene transfer results in stable expression

of hFIX in normal mice

Mice from different strains received IV injections of high-dose

(1 1010

TU/kg) RV as newborns. Expression was stable in allanimals for the duration of evaluation (Figure 1B) and averaged

144 ZHANG et al BLOOD, 1 JANUARY 2004 VOLUME 103, NUMBER 1


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0.4 0.1 g/mL (mean SEM)] hFIX (9% of normal) for C3H,

17.0 7.2g/mLfor BALB/c:129S, 5.5 0.5g/mLfor BALB/c,

and 0.6 0.05 g/mL for C57BL/6 mice. These levels are

therapeutic because more than 10% of normal levels prevent

most bleeding.

Neonatal gene transfer fails to induce antibody formation

in normal mice

Plasma collected from the RV-treated mice was also evaluated for

anti-hFIX antibodies. To determine whether these strains could

produce antibodies, normal mice that did not receive gene transfer

were injected with 30 IU/kg hFIX, which is the dose used for a

minor bleed. The recombinant hFIX used contained an alanine atposition 148 of the mature protein and was identical to that encoded

by the RV. Protein was injected once a week, the frequency at

which a patient with severe HB might be treated. Ten doses were

given because inhibitors usually develop within 8 to 12 days of

exposure.35 hFIX was injected IP because this method is easier than

performing IV injections. Antibody levels were relative amounts in

milligrams per milliliter after comparison with standards in which

mouse IgG was bound to wells followed by incubation with the

anti-mouse IgG antibody. However, the calculated value is not a

correct measure of the amount of anti-hFIX antibody because not

all the protein in the standards binds to the well (L.X., K.P.P.,

unpublished data, July 2001). C3H and BALB/c:129S mice that

received protein injections without preceding gene transfer consis-

tently made very high-titer antibodies to hFIX, with averagerelative IgG levels of 3.8 1.4 and 12.2 5.2 mg/mL, respec-

tively, as shown in Figure 2A and as summarized in Table 1.

Although those BALB/c mice that produced antibodies had high

average relative IgG levels (13.8 4.6 mg/mL), 25% failed to

produce any antibodies. Only 5 of 8 C57BL/6 mice developed

antibodies, and the average peak relative IgG level of 1.7 mg/mL

was lower than that in animals from the other strains.

Although all strains were capable of producing anti-hFIX

antibodies after protein infusion, none of the mice that received

neonatal gene transfer had anti-hFIX antibodies 2 months or later

after birth (Table 1). To further test whether the neonatal gene

transfer approach can induce tolerance, RV-treated mice of the

strains with the most robust antibody response were challengedwith hFIX beginning at 4.5 months after birth. They received 30

IU/kg hFIX, which increases plasma levels to approximately 1.5

g/mL in humans. Given that the pharmacokinetics are similar in

mice,46 this dose should increase blood levels by 3.4-, 0.1-, and

0.3-fold for C3H, BALB/c:129S, and BALB/c mice, respectively.

None of the RV-treated mice developed anti-hFIX antibodies after

10 injections of protein, as shown in Figure 2B for C3H and

BALB/c:129S mice and as summarized in Table 1 for BALB/c

mice. As a final test of the ability of neonatalgene transfer to induce

tolerance, these mice were injected twice with hFIX in adjuvant.

All mice were tolerant as they continued to have stable expression

of hFIX (Figure 1B) without antibody formation (Figure 2B; Table

1). We conclude that neonatal gene transfer with an RV dose that

results in high-level expression induces tolerance to hFIX.

Induction of tolerance in HB mice

The ability of neonatal gene transfer to induce tolerance was also

tested in HB mice that do not express detectable antigen. 44 Because

the human and mouse proteins are 80% identical, immune re-

sponses in null HB mice might differ from those in normal mice.

Neonatal HB mice with a mixed 129SC57BL/6 background were

injected intravenously with high (1 1010 TU/kg), medium

(1 109 TU/kg), low (1 108 TU/kg), or very low (1 107

TU/kg) doses of RV to determine whether expression level affected

the ability to induce tolerance to the transgene. Expression was

stable in most animals from 2 to 8 months after birth, with the

exception of 1 animal in the very low-dose group (to be described

at the end of this section). Average levels for those that maintained

expression were 8204 4993 ng/mL (164% of normal), 251 188

ng/mL (5% of normal), 50 30 ng/mL (1% of normal), and

2.2 0.8 ng/mL (0.04% of normal) for the high, medium, low, and

very low dose of RV, respectively, as shown in Figure 3A.

Sixty-nine percent of control adult HB mice that did not receive

gene transfer developed moderate levels of antibodies (1.2 0.3

Figure 1. Retroviral vector and expression levels in normal mice from different

strains after neonatal transduction. (A) hAAT-hFIX-WPRE. The RV contains intact

LTRs at the 5 and 3 ends, an extended packaging signal (), the 403-nt human

1-antitrypsin promoter (hAAT), the 1.5-kb hFIX cDNA (hFIX), and the 591-nt

woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Transcrip-

tion can initiate from the LTR or hAAT promoters, as indicated by the arrows.

(B) Expression in normal mice from different strains after neonatal transduction. C3H

(N 5), BALB/c:129S (N 5), BALB/c (N 7), or C57BL/6 (N 3) mice were

injected with 1 1010 TU/kg at 2 or 3 days after birth. Average hFIX antigen levels

SEM are shown at the indicated time in months after birth.

Figure 2. Anti-hFIX IgG antibodies in normal mice after protein injection or

neonatalgene transfer. (A)Anti-hFIX antibody levels after protein injections. Miceof

the indicated strain that did not receive gene transfer began to receive weekly IP

injections of 30 IU/kg hFIX at 2 to 4 months after birth, for a total of 10 doses. The

relativelevels of anti-hFIXIgG antibody in milligrams per milliliterwere determined by

immunoassay and are plotted versus the time in weeks after the first dose of protein.

Each line represents a single animal. For the BALB/c and C57BL/6 mice, 2 and 3

mice, respectively, failed to make antibodies (plotted as 0.001 mg/mL on this semilog

scale) at any time of evaluation, as indicated by the N near the line at the bottom.

(B) Anti-hFIX antibody levels in mice after neonatal gene transfer. These are the

same C3H and BALB/c:129S mice that received neonatal injection of 1 1010 TU/kg

of hAAT-hFIX-WPRE as described in Figure 1B. At 4.5 months after transduction,

mice began to receive weekly injections of hFIX without adjuvant for a total of 10

doses, as indicated by the short black arrows. At 7 and 7.75 months after

transduction, mice received hFIX in adjuvant, as indicated by the long open arrows.

Anti-hFIX IgG antibody levels are shown at the indicated time in months after

transduction. Noneof theC3H (N 5) or BALB/c:129S (N 3) micemade detectableantibodies at any time of evaluation.

NEONATAL GENETRANSFER RESULTS IN TOLERANCE FOR HB 145BLOOD, 1 JANUARY 2004 VOLUME 103, NUMBER 1


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relative mg/mL IgG) after 8 to 10 injections of hFIX without

adjuvant (Figure 3B; Table 2). Anti-hFIX antibody formation

occurred in 100% of mice that received 2 doses of hFIX in

adjuvant, and the average levels were 1.8-fold those in animals that

received injections without adjuvant (Figure 3C; Table 2). Approxi-

mately 50% of the HB mice designated to receive 10 doses of hFIX

protein and 25% of those designated to receive 2 doses of hFIX

with adjuvant died because of bleeding complications.At 4.5 months after birth, RV-treated mice began to receive

weekly IP injections of 0.6 IU BeneFix without adjuvant, which

was continued for 10 weeks. This dose should have increased hFIX

plasma levels to 0.2-, 6-, 30-, and 680-fold those of the pre-hFIX

protein injection levels for mice that received the high, medium,

low, and very low doses of RV, respectively. Five of 6 mice that

received the high RV dose had no detectable antibodies, whereas 1

had a very low level (relative IgG, 0.007 mg/mL) of an anti-hFIX

antibody at 4 weeks after the first dose of hFIX that subsequently

disappeared (Figure 3D); this was considered biologically insignifi-

cant. The frequency of antibody formation in mice that received the

high RV dose was statistically lower than it was in HB mice that did

not receive gene transfer but received 8 to 10 hFIX injections

without adjuvant (P .004, Fisher exact test). Similarly, 5 of 6animals that received the medium RV dose had no detectable

antibodies after 10 hFIX injections. One mouse had a low level

(relative IgG, 0.03 mg/mL) at 6 weeks, which fell to barely

detectable levels by 10 weeks and was considered biologically

insignificant (Figure 3E). There was no loss of animals in either

group because of bleeding complications.

For animals that received the low dose of RV, 0 of 7 mice that

completed 8 or 10 doses of hFIX made anti-hFIX antibodies

(P .004 [Fisher exact test] compared with HB mice after 10

hFIX injections without adjuvant), as shown in Figure 3F. Three

mice in this group died early of bleeding complications, suggesting

that a plasma hFIX level of 50 ng/mL (1% of normal) does not

achieve hemostasis. These mice were further challenged with 2injections of hFIX in adjuvant. Of the mice that survived 1 or 2

injections, 1 developed an anti-hFIX antibody of a moderate level

(relative IgG, 0.4 mg/mL), and the others remained negative

(Figure 3F). The frequency of antibody formation after the

administration of hFIX in adjuvant (1 of 4) remained lower than it

was in HB mice that did not receive gene transfer and received

hFIX in adjuvant (P .01, Fisher exact test).

One mouse that received the very low RV dose developed an

anti-FIX antibody with a relative IgG level of 0.21 mg/mL after 10injections of hFIX without adjuvant (Figure 3G); this was associ-

ated with a decrease in plasma hFIX antigen to undetectable levels.

The antibody level increased further after 1 dose of hFIX in

adjuvant. Although antibodies did not develop in the other 2

animals that completed 10 injections of hFIX without adjuvant, the

frequency of antibody formation in this group (1 of 3) was not

statistically different from that in control HB mice that received 8

to 10 hFIX injections without preceding gene transfer. Mice that

did not develop antibodies after injections of protein without

adjuvant were then injected with hFIX with adjuvant. One devel-

oped an antibody, but the other did not survive the first injection.

Because of the low survival rate in this group attributed to bleeding,

additional HB mice were injected with the very low RV dose atbirth. This resulted in average hFIX levels of 1.6 0.6 ng/mL at 6

weeks (data not shown), which was similar to the level observed in

the initial study. One of 18 mice developed an antibody in response

to gene transfer, but the level (less than 0.012 relative mg/mL IgG)

was low. However, all 17 mice that completed 2 injections of hFIX

in adjuvant developed anti-hFIX antibodies, with an average

relative IgG level of 1.25 0.4 mg/mL. We concluded that the

high, medium, and low doses of RV result in tolerance to hFIX

protein injections but that the very low dose does not.

Inhibitor formation in normal and HB mice

Samples with the highest IgG levels were also tested for inhibitor

activity. All C3H and BALB/c:129S mice that did not undergo thepreceding gene transfer and were challenged with hFIX without

Table 1. Summary of anti-hFIX IgG antibody formation in normal mice

Treatment

group and

mouse strain

No. with antibodies* Average IgG,

relative mg/mL

(range) Average ELISA titer (range)

Average

inhibitor titer,

BU/mL (range)ELISA Bethesda assay

Protein injections without gene transfer

C3H 7 of 7 7 of 7 3.8 1.4 (0.5-10.6) 263 314 132 467 (102 400-409 600) 16 2 (6-20)

BALB/c:129S 8 of 8 8 of 8 12.2 5.2 (0.6-34.4) 421 410 169 746 (12 800-1 000 000) 80 30 (12-200)

BALB/c 6 of 8 6 of 8 13.8 4.6 (0.2-25.9) 1 097 600 342 040 (6 400-1 638 400) 92 32 (1.8-180)C57BL/6 5 of 8 2 of 8 1.7 1. 3 ( 0. 04 -6. 7) 24 000 19 622 (1 600-102 400) 9 8 (1 and 18)

High-dose neonatal gene transfer followed by 10 hFIX injections without adjuvant and 2 hFIX injections with adjuvant

C3H 0 of 5 P .0008 0 of 5 0 1:100 1

BALB/c:129S 0 of 3 P .006 0 of 3 0 1:100 1

BALB/c 0 of 7 P .007 0 of 7 0 1:100 1

High-dose neonatal gene transfer without protein injections#

C57BL/6 0 of 3 NS 0 of 3 0 1:100 1

*Number of animals with significant anti-hFIX IgG antibodies was determined from the total number of animals evaluated. P values were obtained by comparing the

frequency of antibody formationusing Fisher exacttest foranimalsthat receivedgene transferwith thatin miceof the samestrainthat did not receive genetransfer butreceived

10 injections of hFIX without adjuvant.

Average relative levels of anti-hFIX IgG SEM were determined using the highest value obtained for each animal with detectable antibodies.

Average titer was determined using the highest value for each animal that was positive.

Bethesda titer was determined for the sample with the highest levels of anti-hFIX IgG antibody in the immunoassay.

Results aregiven foranimalsof theindicatedstrain that didnot receive gene transferand were treated with 10injectionsof 30IU/kgperdosehFIXwithout adjuvant.These

are the same mice whose antibody levels are shown in Figure 2A.

Results are given for mice of the indicated strain that were injected with 1 1010 TU/kg hAAT-hFIX-WPRE at birth and then received 10 injections of 30 IU/kg per dose

hFIX without adjuvantfollowedby 2 injections of 30 IU/kg hFIX with adjuvant.These arethe same mice whose expression andantibody levelsare shownin Figures 1B and2B,

respectively.

#Results are given for mice of the indicated strain that were injected with 1 1010 TU/kg hAAT-hFIX-WPRE at birth and were not challenged with protein. These are the

same mice whose expression levels are shown in Figure 1B.

NS indicates not significant.



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adjuvant developed inhibitors, which correlated reasonably well

with the relative levels of anti-hFIX IgG (Figure 4A-B; Table 1).

Similarly, all BALB/c mice with anti-hFIX antibodies detectable

by immunoassay had inhibitors (Figure 4C), though those that were

negative by immunoassay were also negative by the Bethesda

assay (data not shown). Inhibitor titers were generally low orundetectable for the C57BL/6 mice (Figure 4D). Inhibitor levels

were similar for HB mice that did not receive gene transfer

regardless of whether they were stimulated with hFIX with or

without adjuvant (Figure 4E).

Inhibitors were also evaluated in mice that received neonatal

gene transfer. None of the C3H, BALB/c:129S, or BALB/c mice

treated with gene transfer at birth and challenged with hFIX

developed inhibitors (Table 1), which is consistent with the absence

of anti-hFIX antibodies by immunoassay. Similarly, none of the

C57BL/6 mice developed inhibitors, though protein stimulation

was not performed. Inhibitors were also absent from all HB mice

that received the high, medium, or low dose of RV and were

challenged with protein (Table 2). However, most mice that

received the very low dose of RV developed inhibitors in response

to protein administration (Figure 4F; Table 2). We conclude that

administering a higher dose of RV to newborns results in tolerance

to protein infusion but that administering the very low dose

does not.

Neonatal gene transfer in normal dogs

Five normal dogs were injected with hAAT-hFIX-WPRE at 2 or 3

days after bir th. T he platelet counts wer e nor mal at

183 000 26 000 and 175 000 104 000 at 24 and 48 hours after

injection, respectively, which suggests that the modest decrease in

the platelet count noted previously with a 3-fold higher dose of

RV11 was dose related. All dogs had stable expression of hFIX,

which varied from 223 to 914 ng/mL in individual animals and

averaged 494 132 ng/mL (Figure 5A). No animals developed

anti-hFIX antibodies as assessed by immunoassay or Bethesda

assay (Figure 5C; Table 3).

Two experiments documented that this colony of dogs could

produce anti-hFIX antibodies. Three dogs injected with hAAT-hFIX-

WPRE at 8 to 11 weeks after birth exhibited low-level expression at

1 week (Figure 5B), which averaged 14.3 6.3 ng/mL. Two dogs

had subsequent decreases in their plasma hFIX antigen levels inconjunction with the development of anti-hFIX antibodies that

were of relatively low titer (Figure 5D), whereas the third dog

maintained hFIX levels at approximately 8 ng/mL for 6 months and

never developed anti-hFIX antibodies. In addition, 2 dogs injected

intravenously with 10 doses of 30 IU/kg hFIX starting at 8 weeks

after birth developed high-titer anti-hFIX antibodies (Figure 5E)

with Bethesda titers of 2 and 5 BU/mL (Table 3). A third dog

developed a low-titer antibody without inhibitory activity that

disappeared with time.

DiscussionNeonatal gene transfer does not induce anti-hFIX antibodies

in mice or dogs

This study demonstrates that neonatal gene transfer with a high

dose (1 1010 TU/kg) of an amphotropic RV expressing hFIX

does not induce anti-hFIX antibody formation in C3H, BALB/c:

129S, BALB/c, C57BL/6, or HB mice. In contrast, mice from these

strains produce anti-hFIX antibodies after protein infusion as

adults, albeit with varying efficiency. Similarly, none of 5 dogs that

received neonatal gene transfer with hAAT-hFIX-WPRE devel-

oped antibodies, though clinically significant anti-hFIX antibodies

developed after protein infusion in 2 of 3 normal dogs in this study

and in 6 of 8 normal47

and 6 of 6 HB48

dogs in previous studies. Thefrequency of anti-hFIX antibody formation in dogs is statistically

Figure 3. hFIXexpressionand anti-hFIX IgGantibody levels in HB mice. (A) hFIX

levels in mice transduced as neonates. Neonatal 129SC57BL/6 HB mice were

injected IV with a high (1 1010 TU/kg), medium (1 109 TU/kg), low (1 108

TU/kg), or very low (1 107 TU/kg) dose of hAAT-hFIX-WPRE at 2 or 3 days after

birth. Average plasma hFIX antigen levels SEM are shown. (B) Anti-hFIX IgG

antibody levels after hFIX protein injection. Adult HB mice that never received gene

transferbegan toreceiveweekly IPinjectionsof 30IU/kghFIXwithoutadjuvant at2 to

4 months after birth and continued for 10 injections total, as indicated by the short

vertical arrows in this and subsequent panels. Plasma anti-hFIX antibody levels weredetermined at the indicated time in weeks after the first dose of hFIX. Each line

indicates an individual mouse. Values are plotted as 0.001 mg/mL for the 5 mice that

failed to make antibodies at any time of evaluation. (C) Anti-hFIX IgG antibody levels

after hFIX protein injection with adjuvant. Adult HB mice that never received gene

transfer received 2 injections of 30 IU/kg hFIX in adjuvant separated by 3 weeks.

Long open arrows indicate the time of injection of protein with adjuvant in this and

subsequent panels. Plasma anti-hFIX IgG antibody levels are plotted versus the time

after the first dose of hFIX. Each line indicates an individual mouse. (D-H) Anti-hFIX

IgG antibody levels in HB mice that were transduced as neonates. Plasma from mice

that were treated at birth with a high (D), medium (E), low (F), or very low (G-H) dose

of hAAT-hFIX-WPRE and began to receive hFIXproteininjections at 4.5 months after

birth was tested for anti-hFIXspecific IgG antibodies at the indicated time after birth.

These are the same animals whose hFIX levels are shown in panel A. For panels D

and E, the line with open circles represents an individual mouse with low and

transient levels of an antibody, whereas the line with closed circles represents 5 mice

that did not have detectable antibodies at any time of evaluation. For panel F, the line

with open circles represents an animal that developed an antibody after administra-

tion of1 dose ofhFIXin adjuvant.Thelinewithclosed trianglesrepresents3 mice thatdid not develop antibodies after 10 injections of hFIX without adjuvant. The line with

closed circles represents 3 mice that did not develop antibodies after 10 injections of

hFIX without adjuvant and 1 or 2 injections of hFIX with adjuvant. (G) Anti-hFIX IgG

antibody levels in HB mice that were transduced with the very low dose of RV as

neonates and were challenged with hFIX as indicated. Each line represents an

individual animal. Neonatal mice were injected at birth with the very low dose of RV

and were challenged at 2 and 2.75 months with hFIX in adjuvant. Each line indicates

an individual animal.



6/9

lower after neonatal gene transfer than after protein infusion if

these historical controls are included (P .002, Fisher exact test).

These results are consistent with our previous study in which

significant levels of anti-cFIX antibodies did not develop after

neonatal transfer of an RV expressing cFIX to mice and dogs11 and

the absence of anti-hFIX antibodies after neonatal gene transfer

with AAV49 or adenoviral46 vectors. Our results differ from those ofVandenDrissche et al,50 who found inhibitors in 50% of hemophilia

A mice that received neonatal IV injections of a VSV-G

pseudotyped RV expressing human factor VIII (hFVIII). This

discrepancy could be attributed to a greater immunogenicity of

hFVIII, induction of inflammatory responses by VSV-G or to

other causes.

Although others have suggested that liver-restricted expression

can reduce or prevent an antibody response after gene transfer with

AAV3,4,18 or adenoviral19,20 vectors, it is unlikely that this is the

mechanism here. We previously found that expression was high in

the spleen from the long terminal repeat (LTR) of an RV at 5 days

after neonatal transfer in dogs,41 and the LTR of our RV (Figure 1A)

can direct expression of hFIX in nonhepatic cells. In addition,spleen mRNA levels were approximately 1% those in liver at 6

months after neonatal injection of a similar vector into mice.51

Studies are in progress to confirm that expression occurs in the

spleen shortly after neonatal gene transfer in mice.

Anti-hFIX antibodies were still not observed in most mice after

neonatal gene transfer with progressively lower doses (1 109,

1 108, or 1 107 TU/kg) of hAAT-hFIX-WPRE in HB mice.

This result differs from that of Mingozzi et al,18 who reported thatlower expression of the transgene after an AAV vector was

delivered to the livers of adult mice was more likely to result in

antibody formation to FIX than was higher expression. Possible

explanations for this discrepancy include differences in the ages or

the genetic backgrounds of the mice.

Neonatal gene transfer induces dose-dependent

tolerance to hFIX

Mice that received neonatal injections of hAAT-hFIX-WPRE were

tested for tolerance to hFIX for 2 reasons. First, some patients will

probably not achieve fully therapeutic levels of hFIX after gene

transfer and would have to be treated intermittently with factor.Second, low-dose neonatal gene therapy might be used to induce

Table 2. Summary of anti-hFIX IgG antibody formation in HB mice

Treatment group No. with antibodies*Average anti-hFIX

IgG, mg/mL

(range)

Average anti-hFIX

antibody titer

(range)

Average

inhibitor titer,

BU/mL (range)Dose of RV

hFIX protein

injections ELISA

Bethesda

assay

hFIX protein injections into HB mice that did not receive gene transfer

None 8-10 without adjuvant

(HB 10)

11 of 15 9 of 15 1.2 0.3

(0.05-3.2)

20 436 6 140

(800-51 200)

7.5 2.2

(1.6-18)

None 2 with adjuvant

(HB 2)

10 of 10 10 of 10 2.1 0.5

(0.6-4.6)

143 000 34 800

(40 000-320 000)

9.1 2.2

(1-18)

Neonatal gene transfer to HB mice before hFIX protein injections

High-dose RV, 1 1010 TU/kg 10 without adjuvant 0 of 6

P .004 vs

HB 10

0 of 6 0 1:100 1

Medium-dose RV, 1 109 TU/kg 10 without adjuvant 0 of 6

P .004 vs

HB 10

0 of 6 0 1:100 1

Low-dose RV, 1 108 TU/kg None 0 of 10 0 of 10 0 1:100 1

10 without adjuvant 0 of 7

P .004 vs

HB 10

0 of 7 0 1:100 1

10 without adjuvant

and 1-2 with

adjuvant

1 of 4

P .01 vs

HB 2

0 of 4 0.4 3 200 1

Very-low-dose RV, 1 107 TU/kg None 2 of 24 0 of 24 0.009 0.003

(0.006 and 0.012)

300 100

(200 and 400)

1

10 without adjuvant 1 of 3

NS vs

HB 10

0 of 3 0.21 6 400 1

10 without adjuvant

and 1-2 with

adjuvant

2 of 2

NS vs

HB 2

1 of 2 0.355 0.345

(0.01-0.7)

3 400 3 000

(400 and 6 400)

18

2 with adjuvant 17 of 17

NS vs

HB 2

17 of 17 1.25 0.4

(0.06-5.8)

61 412 18 017

(2 000-200 000)

21.9 10.3

(1.8-180)

*Number of animals with significant anti-hFIX IgG antibodies was determined out of the total number of animals evaluated. Pvalues were obtained by comparing the

frequency of antibody formation using Fisher exact test for animals that received gene transfer with that in HB mice that received 10 injections of hFIX without adjuvant (HB 10)

or 2 injections of hFIX with adjuvant (HB 2).Average relative levels of anti-hFIX IgG SEM were determined using the highest value obtained for each animal with detectable antibodies.

Average anti-hFIX IgG titer was determined using the highest value for each animal that was positive.

Bethesda titer was determined for the sample with the highest anti-hFIX IgG antibody level and was averaged for all the animals that were positive.

Results are given for HB mice that did not receive gene transfer and were treated with 10 injections of 30 IU/kg per dose hFIX without adjuvant (HB 10) or 2 doses of

30 IU/kg hFIX with adjuvant (HB 2). These are the same mice whose antibody levels are shown in Figure 3B-C, respectively.

Results are given for HB mice that were injected with different doses of hAAT-hFIX-WPRE at birth. These are the same mice whose expression and antibody levels are

shown in Figure 3A, D-H, respectively.



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tolerance to factor infusion, which should have a proportionately

lower risk for adverse effects. C3H, BALB/c:129S, and BALB/c

mice that received a high dose (1 1010 TU/kg) of RV failed to

develop anti-hFIX IgG antibodies after 10 IP injections of hFIX

protein without adjuvant. It is possible that different results wouldhave been obtained with IV injections, which is the route used in

humans. However, this is unlikely because most proteins rapidly

reach the blood after IP injection, and that was used in this study

because it is easier to perform. These mice also failed to make

antibodies after injections of 2 doses of hFIX in adjuvant, which

is a more stringent test of tolerance. Similarly, HB mice that

received a high or medium (1 109 TU/kg) dose of RV failed to

develop anti-hFIX antibodies in response to 10 injections of

hFIX without adjuvant.

In contrast to the results with the high and medium doses of RV,

some HB mice that received lower doses of RV as newborns

developed antibodies after challenge with hFIX. For the low-dose

(1 108 TU/kg) group, the frequency was statistically different

from that in HB mice that did not receive gene transfer. Thus,although induction of tolerance was incomplete, it was still

markedly reduced, and the antibody that developed was of low titer.

For the very low dose (1 107 TU/kg), the frequency of antibody

formation with simple protein injection was harder to assess, given

the small number of animals that survived, because of bleeding, but

it was not statistically different from that in HB mice that did not

receive gene transfer before protein challenge. All animals that

received the very low RV dose developed antibodies after hFIX

injection with adjuvant.

We conclude that continuous expression of more than 14 ng/mL

(3 1010 M) hFIX starting shortly after birth results in tolerance

to the administration of protein in adulthood. This is consistent with

the observed tolerance in transgenic mice that express antigen at108 to 1010 M, though lower expression was insufficient to

induce tolerance.52-60 In these studies of transgenic mice, the

absence of antibodies in vivo is attributed to T-cell tolerance; B

cells remain capable of responding when incubated with T cells

from nontransgenic mice. Future studies will determine whether

the induction of tolerance after neonatal RV gene transfer is caused

by a similar mechanism.

Implications for patients with hemophilia

Neonatal gene therapy might be used to reduce bleeding in patients

with HB if long-term preclinical data demonstrate safety. These

data suggest that this neonatal RV-mediated gene therapy approach

will not induce antibody formation, regardless of the expression

level. However, the immune system of newborn humans is

relatively more mature than that of newborn mice, though immune

responses in newborn humans are still markedly blunted relative to

that of adult humans.37,38 It will, therefore, be necessary to confirm

in future studies that neonatal gene therapy does not induce

immune responses in large animals, including primates, before this

approach is used in humans with HB.

One use for neonatal gene therapy for hemophilia would be to

induce tolerance to the subsequent infusion of protein with arelatively low dose of RV that should have a reduced risk for

adverse effects. Although inhibitors develop in only 3% of patients

with HB, they occur in 35% of patients with hemophilia A with

large deletions or early truncations.61 Future studies will determine

whether the expression of more than 3 1010 M hFIX induces

tolerance in larger animals and whether tolerance to hFVIII occurs.

Implementing this approach for inducing tolerance in patients will

also require long-term evaluation of the safety of neonatal

gene transfer.

Figure 4. Inhibitor formation in normal and HB mice. The inhibitor activity for the

sample from each mouse with the highest antibody level is plotted versus the

anti-hFIX IgG level for that sample. (A-D) Values are shown for mice of the indicated

strain that did not receive gene transfer and were challenged with 10 doses of 30 IU

hFIX without adjuvant. (E) HB mice that did not receive gene transfer were

challenged with 10 injections of 30 IU/kg hFIX without adjuvant (F) or 2 doses of 30

IU/kg hFIX with adjuvant (E). (F) HB mice were treated with the very low dose of RV.

One mouse (the time course of antibody levels for this mouse is shown as a E in

Figure3G)was challenged with 10dosesof hFIX without adjuvantand 1 dose ofhFIX

with adjuvant (). The other mice (shown in Figure 3H) were stimulated with 2 doses

of hFIX with adjuvant and are shown as E here.

Figure 5. Expression of hFIX and anti-hFIX IgG levels in normal dogs after gene

transfer or protein injection. (A) hFIX antigen levels after neonatal gene transfer.

Newborn normal dogs (N 5) were injected intravenously with 3.2 109 TU/kg

hAAT-hFIX-WPRE at 2 days after birth, and plasma was tested for hFIX antigen

levels at the indicated time in months after transduction. (B) hFIX antigen levels after

gene transfer to young dogs. Two 8-week-old dogs (B85 and B90) were injected

intravenously with 5 108 TU/kg hAAT-hFIX-WPRE, whereas one 11-week-old dog

(M1595) was injected intravenously with 2 108 TU/kg. The plasma was tested for

hFIX antigen levels at the indicated time in months after transduction. Antigen levels

that were undetectable were plotted as 0.5 ng/mL on this semilog scale. (C-E)

Anti-hFIXIgG antibody levels in dogs. Anti-hFIXIgG antibody levels weredetermined

by immunoassay. If antibody was undetectable, it was plotted as 0.1 g/mL on this

semilog scale. (C) Plasma was from the dogs that were transduced as newborns and

whose antigen levels are shown in panel A. Time of evaluation varied from 6 to 9

months after birth. (D) Plasma was from the dogs that were transduced as juveniles

and whose antigen levels are shown in panel B, and the values are plotted at the

indicated times after transduction. (E) Plasma was from dogs that began to receive

weekly IV injections of 30 IU/kg hFIX at 8 weeks after birth, which was continued for

10 weeks, as indicated by the black arrows. Antibody levels are plotted versus thetime after the first dose of hFIX protein.



8/9

A final implication of this study is that patients might be tolerized to

hFIX (or hFVIII) by achieving a relatively stable level of protein in

blood with frequent protein injections during the first several months

after birth. Indeed, injecting hFVIII into newborn mice resulted in

tolerization to protein challenge when they became adults,62 whereas

initiating frequent injections of hFIX at birth led to the development of

tolerance in HB dogs from Chapel Hill.63 These results provide a

rationale for testing whether frequent administration of factor immedi-

ately after birth can reduce the frequency of inhibitor formation in

patients at high risk for their development.

Acknowledgments

We thank Donna Armentano and Savio Woo for the modified hFIX

cDNA, Wyeth Pharmaceutical for BeneFix, Hui-Feng Lin and

Darrel Stafford for HB mice, Paul Monahan and Chris Walsh for a

canine anti-hFIX antibody, Roland Herzog for advice on immuno-

assays, and Patty ODonnell and Karyn Cullen for assistance with

dog studies.

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Table 3. Summary of anti-hFIX IgG antibody formation in dogs

Treatment group

Dogs with

antibodies*

Identifying

no.

Peak relative

IgG, g/mL

Peak immunoassay

titer

Inhibitor titer,

BU/mL

Dogs transduced at birth 0 of 5 0 1:100 1

Dogs transduced at 8-11 wk 2 of 3 M1595 0 1:100 1

B85 41 1:800 1

B90 19 1:400 1

Dogs injected with 10 doses hFIX protein starting at 8 wk 3 of 3 M1641 631 1:102 400 2M1644 252 1:25 600 5

M1645 10 1:200 1

*Number of animals with antibodies was determined out of the total number evaluated.

Bethesda titer was determined for the sample with the highest levels of anti-hFIX IgG or for the sample collected at the last time point.

Dogs were injected with 3.2 109 TU/kg hAAT-hFIX-WPRE at 2 or 3 days after birth and were never stimulated with hFIX protein injections. These are the same dogs

whose hFIX antigen and anti-hFIX antibody levels are shown in Figure 5A,C.

Dogs were injected with 5 108 TU/kg hAAT-hFIX-WPRE at 8 weeks after birth (B85 and B90) or 2 108 TU/kg hAAT-hFIX-WPRE at 11 weeks after birth (M1595) and

were never stimulated with hFIX protein. These are the same dogs whose hFIX antigen and anti-hFIX antibody levels are shown in Figure 5B,D.

Dogs that did not receive gene transfer were injected intravenously with 10 doses of 30 IU/kg hFIX beginning at 8 weeks after birth. These are the same dogs whose

anti-hFIX antibody levels are shown in Figure 5E.



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